Our long-term goal is to understanding RNA editing in trypanosome, very primitive eukaryotes that cause disease in tens of millions plus major economic hardship, and for which effective treatment is lacking. Trypanosome RNA editing is one of the strangest known forms of RNA maturation, where U residues are inserted into primary mitochondrial transcripts (often on a massive sale) and less frequently deleted from these RNAs, to create the mature coding regions. This editing is directed by base pairing with guide RNAs, and only recently it has been shown to be catalyzed by protein enzymes. We have isolated a complex of seven polypeptides that catalyzes both U-deletional and U-insertional editing and co-purifies with the implicated component activities: gRNA-directed endonuclease, 3'-U exonuclease, terminal-U-transferase, and RNA ligase. [Two of these polypeptides are RNA ligase, so likely all the activities are directed by these seven polypeptides.] We propose several routes to pursue RNA editing. In a biochemical approach to understand how this simple editing complex functions, we will determine what mRNA and gRNA features specify U-insertion, U-deletion, and their cleavage reactions and will follow up an indication of an accessory factor that aids this recognition. We will also pursue other features that we found distinguish U-insertion and U-deletion, including their inverse allosteric control by adenosine nucleotides and a selective inhibition by added cloned edition polypeptides II and III, and we will examine how the editing complex recognize and then acts on its substrate RNAs. We also propose a major effort studying the seven polypeptides using recombinant techniques. We have already cloned five of these genes and intend to clone the other two, and will examine the role of their products by in vitro expression, genetic knock-outs, and in vivo and in vitro reconstitution studies, including the use of mutated genes. We will also pursue the regulation and internal organization of these polypeptides. Finally, we will examine additional roles of the editing polypeptides, in particular the suggestion that polypeptide IV may be the next poly-A-polymerase that generates the 3' end of mitochondrial mRNAs, and we will attempt to develop next generation in vitro systems that catalyze more than one editing cycle and use sequential guide RnAs. We feel that our advances during the last granting period have put us in a very good position to make considerable progress in understanding the mechanism of this unique form of RNA maturation.